Conditions for maintenance of embryonic chick duodenum in organ culture in serum-free medium, responsive to vitamin D and many analogues, with excellent preservation of mucosal structure, have been essentially optimized. It is proposed to further investigate the regulation of synthesis and physiological role(s) of the vitamin D3-induced calcium-binding protein (CaBP) in the calcium absorptive mechanism under the precisely controllable in vitro conditions, completely isolated from systemic influences, made possible only through the use of several variations of this unique system. Experiments to date with this technique have established that vitamin D itself (and numerous analogues) induces de novo synthesis of CaBP and stimulates transmucosal calcium transport by a calcium-dependent mechanism involving the adenyl cyclase system. Reconstitution experiments with purified CaBP have proven the involvement of this protein in the calcium transport process. Considerable further investigation of the interaction between vitamin D, CaBP, calcium and cyclic AMP will be undertaken. In this regard attempts will be made to localize and characterize hypothetical vitamin D receptors of the adenyl cyclase system. The effects of a variety of agents, known to influence calcium absorption in vivo, will be investigated. Among these are cortisone, recently shown to interact with vitamin D3 in CaBP biosynthesis and function in this system, diphenylhydantoin, shown to directly inhibit the intestinal calcium absorptive mechanism, and other physiological and pharmacological agents, including several carcinogens, one of which was recently shown to interfere with CaBP biosynthesis. Attempts will be made to isolate, and use in reconstitution experiments, CaBP-mRNA. Finally, recent work with synthetic alpha,25-dihydroxy vitamin D3 indicates that this system could be used as a highly sensitive, specific and relatively simple bioassay for this biologically significant steroid. BIBLIOGRAPHIC REFERENCES: R.A. Corradino, Diphenylhydantoin: direct inhibition of the vitamin D3-mediated calcium absorptive mechanism in organ-cultured duodenum. Biochemical Pharmacology 25 (1976),(In press). R.A. Corradino, Embryonic chick intestine in organ culture: Earliest action of alpha,25-(OH)2D3 is the stimulation of cyclic AMP production. (Abstract). (Text Truncated - Exceeds Capacity)